The goal of this research project is to understand the nature of mouse embryonic and adult stem cells and to identify genes that are responsible for the maintenance of cellular pluripotency. We have been conducting global gene expression profiling with the mouse embryonic cDNA microarrays developed in our laboratory. As a first step, large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had previously been characterized, the majority (67%) of the ES-specific genes was novel and did not include known differentiated cell markers. We suggest that pluripotency requires a set of genes not expressed in other cell types, while lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage. The identification of genes that are specifically expressed in ES cells has provided an important first step for understanding the pluripotency of stem cells. These genes can be used as markers for pluripotent stem cells. Furthermore, the manipulation of these genes in ES cells and other cell types can further elucidate their function and provide possible means to harness the cellular pluripotency. To extend this work and extract the common features of stem cells, we have thus far profiled the following stem cells: (1) Differentiation and lineage commitment of pluripotent mouse embryonic stem (ES) cells. Expression profiling of mouse ES cells at six time points during the course of their differentiation has been performed. (2) Mesenchymal stem cells and derivative osteoblast cells. (3) Neural Stem cells and neuron/glia cells. We are currently working on data analysis and independent validation of results. We also plan to do expression profiling on Embryonic Germ (EG) cells, on ES cells with the altered expression of Stat3, and on ES cells with altered expression of Oct-3/4.